Nanopore-matched protein shuttle for molecular characterization and methodology for data analysis thereof

Abstract

Systems and methods are provided for characterizing shuttle capture events in a nanopore sensor. The method first collects time-dependent current blockage signatures for at least one bias voltage. The method then identifies each signature as corresponding to a permanent or transient event. The method then generates a protein dynamics landscape (PDL) for the transient event signatures. The PDL comprises a set of histograms of nanopore current data and characterizes current through the nanopore during shuttle capture events. The method can then comprise identifying an entrance level blockage value based on the permanent event signatures. Permanent event captures can be determined by time duration which is larger than a certain threshold time value. Applying a between the fluidic chambers above a threshold voltage level can be used to control that the vast majority of events are permanent.

What is claimed is: 
     
       1. A method for characterizing shuttle capture in a nanopore sensor, comprising:
 collecting a plurality of time-dependent current blockage signatures for a plurality of different bias voltages; 
 classifying the plurality of time-dependent current blockage signatures into permanent event signatures and transient event signatures; 
 for each of the plurality of bias voltages, generating a protein dynamics landscape (PDL) for the transient event signatures, wherein the PDL comprises a collection of blockage spectra, wherein each of the collection of blockage spectra comprises a histogram of blockage values for events of a selected maximum duration; 
 identifying a permanent level blockage value based on the permanent event signatures; 
 selecting one of the plurality of bias voltages corresponding to a PDL for the transient event signatures where the collection of blockage spectra reveals peaks at blockage values that are substantially equal to the permanent level blockage value. 
 
     
     
       2. The method of  claim 1 , wherein the time-dependent current blockage signature comprises a measurement of nanopore current as a function of time during a shuttle capture event. 
     
     
       3. The method of  claim 1 , wherein the classifying further comprises identifying as the transient event signatures any one of the plurality of time-dependent current blockage signatures showing a return to an initial current level. 
     
     
       4. The method of  claim 3 , wherein the classifying further comprises identifying as the permanent event signatures any one of the plurality of time-dependent current blockage signatures failing to show a return to an initial current level. 
     
     
       5. The method of  claim 4 , further comprising:
 for each of the plurality of bias voltages, calculating a ratio of a number of permanent shuttle capture events to a number of transient shuttle capture events; and 
 based on the calculated ratios, determining a threshold bias voltage to achieve a preferred ratio. 
 
     
     
       6. The method of  claim 1 , wherein the blockage spectra further comprises an average of nanopore current blockage data, sampled over a fixed time interval to yield histogram data points. 
     
     
       7. The method of  claim 6 , wherein the fixed time interval is at least as large as an interval for sampling the nanopore current blockage data. 
     
     
       8. The method of  claim 6 , wherein the nanopore current blockage data comprises a ratio of a change in the current from an input current to the input current. 
     
     
       9. The method of  claim 1 , wherein the identifying comprises:
 generating an additional PDL for the permanent event signatures for at least one of the plurality of bias voltages; and 
 selecting an entrance level blockage value to correspond to a blockage level lower than the permanent blockage level. 
 
     
     
       10. The method of  claim 9 , wherein the method further comprises:
 fitting each spectrum of the collection of blockage spectra within a PDL with a sum of Gaussian distribution; 
 determining the entrance level blockage value from a peak in the collection of blockage spectra within the PDL for permanent events. 
 
     
     
       11. The method of  claim 1 , wherein the selected maximum duration of the particular blockage spectra is at least one of: 2 milliseconds, 5 milliseconds, 10 milliseconds, 30 milliseconds, 100 milliseconds, 500 milliseconds, and 1000 milliseconds.